Phosphorylation of atrial natriuretic peptide prohormone

Previously they have shown that atrial natriuretic peptides (ANP) are excellent substrates for cAMP-dependent protein kinase. The site of in vitro phosphorylation occurs at Ser 104, and is contained in a typical recognition sequence for cAMP-dependent protein kinase, Arg 101-Arg 102-Ser 103-Ser 104. In this report the prohormone pro-ANP, the predominant form present in atrial secretory granules, was purified from rat atria. Like ANP, pro-ANP was also found to be phosphorylated by cAMP-dependent protein kinase in vitro. Peptide mapping studies carried out with in vitro-phosphorylated pro-ANP revealed predominantly one 32P-labeled peptide. This was demonstrated to be the same hexapeptide, Arg 101-Phe 106, found earlier for phosphorylated ANP. The amino acid sequence analysis also suggests that the site of phosphorylation is located at Ser 104. When isolated rat atria were incubated in the presence of 32P-orthophosphate, the pro-ANP purified from these atria was observed to be radioactive. The in situ incorporation of 32P into pro-ANP was confirmed by SDS polyacrylamide gel electrophoresis followed by immunoblotting and autoradiography